A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the brain of the cell that allows it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. In this research project, we have developed and begun applying an entirely new approach to studying the molecular mechanisms of transcription and transcription regulation in vitro. Instead of studying populations of molecules, we directly visualize the RNA polymerase and regulatory proteins on an isolated single DNA molecule, following the progression of the molecular machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by a multi-wavelength single-molecule fluorescence approach we call CoSMoS (co localization single- molecule spectroscopy). In this application, we propose applying the CoSMoS approach to elucidating the dynamic mechanisms of selected processes involved in regulation of transcription initiation and elongation in vitro using purified Escherichia coli proteins in reconstituted transcription reactions. Our goals are: 1) Test the hypothesis that RNAP holoenzyme molecules reach promoters through a bind-and-slide mechanism; 2) Define the dynamic mechanisms by which secondary channel factors act alone and together to regulate transcription initiation and elongation; and 3) Define the dynamic mechanism by which the presence of the 70 and NusA components of the transcription apparatus is remodeled in the transition between transcription initiation and elongation.